CN1268572A - Method for synthesizing 5-enol pyruvoyl-shikimic acid-3-phosphoric acid synthetase gene - Google Patents
Method for synthesizing 5-enol pyruvoyl-shikimic acid-3-phosphoric acid synthetase gene Download PDFInfo
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- CN1268572A CN1268572A CN 99103472 CN99103472A CN1268572A CN 1268572 A CN1268572 A CN 1268572A CN 99103472 CN99103472 CN 99103472 CN 99103472 A CN99103472 A CN 99103472A CN 1268572 A CN1268572 A CN 1268572A
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Abstract
The synthesis of 5-enol pyruvoyl-shikimic acid-3-phosphoric acid synthesis enzyme (EPSPS) gene includes designing synthesis of gene, gene sequence and gene clone. The synthesized gene can be applied in formulating dicotyledon capable of anti glyphosate such as rape, soybean cotton etc and monocotyledon capable of anti glyphosate such as paddy rice corn, wheat etc.
Description
The present invention relates to a kind of method for synthesizing gene, relate in particular to the method for synthetic genes such as the monocotyledons of a kind of dicotyledons that can be applicable to formulate resistance glyphosate such as rape, soybean, cotton etc. and resistance glyphosate such as paddy rice, corn, wheat.
(5-enolpyruvyl-shikimate-3-phosphate synthase EPSPS) is die aromatischen Aminosaeuren to 5-enol pyruvoyl-oxalic acid-3-phosphate synthase, and the key enzyme of tyrosine, tryptophane, phenylalanine building-up process is present in plant and the bacterium.Glyphosate is a kind of wide spectrum, interior suction, steriland herbicide efficiently, can transport by the phloem of plant.It by with the combining of this enzyme molecule, block these amino acid whose biosynthesizing, thereby cause necrocytosis.
Thereby can hinder glyphosate by change EPSPS albumen primary structure and make the resistance of its acquisition glyphosate with combining of enzyme molecule.Last 101 proline(Pro) of Salmonella aroA structure gene (coding EPSPS) sports the resistance (Comai that has obtained behind the Serine glyphosate, L., Sen, L.C., Stalker, D.M., 1983.An altered aroA gene product confers resistanceto the herbicide glyphosate.Science 221:370-371).Learn that from existing result of study at present used resistance glyphosate EPSPS gene all directly clones to come out to transfer in the vegetable cell by molecular biology method again from plant (comprising rape, petunia, cotton, soybean etc.) or bacterium (comprising intestinal bacteria and Salmonella).As, EPSPS gene from Salmonella has imported tobacco (Comai, Letal.1985. Expression in plants of amutant aroA gene from Salmonella typhimuriumconfers tolerance to glyphosate.Nature 317:741-744.), imported tobacco (Della-Cioppa from colibacillary EPSPS gene, G.et al.1987.Targeting a herbicide-resistant enzyme from E.coli to chloroplasts ofhigher plants.Bio/Technology 5:579-584.), EPSPS gene from petunia has imported petunia (Shah, D.M et al.1986.Engineering herbicide tolerance in transgenicplants.Science 233:478-481.).U.S. Meng Shan (Monsanto) company directly clones the EPSPS gene from the variation plant cell of resistance glyphosate, to breed soybean, cotton, the new rape variety of resistance glyphosate in this EPSPS gene transfered plant by transgenic technology, and with this art applications the patent (patent No.: 5188642).
For the present invention, above-mentioned method of directly cloning the EPSPS gene from organism has 2 deficiencies:
1, step is many, program complexity, length consuming time.The method of clone gene is a lot of from organism (especially plant materials) at present, as transposon tagging technology, map based cloning technology etc., but the sort of technology no matter, the required time is all very long, and step is quite loaded down with trivial details.Generally speaking, clone a required time of gene and take 2-3, and cost is also quite huge.
2, gene itself does not have codon optimization, thereby gene expression efficiency is restricted.Usually, gene is one section dna sequence dna that function is arranged, and this segment DNA sequence is exactly molecular by password.Gene at first is transcribed into messenger RNA(mRNA) (being mRNA) in cell, translate into protein again by mRNA then.In this process, rrna at first will be discerned mRNA, starts translating of codon then and reads.Therefore, do not have a preference for if codon is not this biology, then ribosomal recognition efficiency just reduces, and therefore has influence on this expression of gene level.
The objective of the invention is to: the problem at prior art exists provides a kind of quick, economical, the EPSPS gene design synthetic method that expression efficiency is high.At directly 2 deficiencies of clone gene from organism of in background technology, being spoken of, utilize the synthetic EPSPS gene of present method can big time saver, generally only need about 2 months, cost also reduces greatly; Simultaneously, because, can estimate that institute's synthetic EPSPS gene will have higher expression efficiency through optimization to codon.
The present invention is achieved in that the present invention mainly introduces a kind of method principle of artificial design EPSPS gene completely newly and gene and the gene order that obtains with this method.Institute's synthetic EPSPS gene is a heterozygote gene, has the feature of resistance glyphosate.Its coded albumen merges with the proper reading frame frame mutually with signal peptide, and the latter can import to the former in the chloroplast(id) of vegetable cell.The synthetic EPSPS of institute gene can be expressed in vegetable cell and regeneration plant, thereby makes vegetable cell and regeneration plant obtain resistance to glyphosate.Described plant comprises monocotyledons such as paddy rice, corn, wheat etc., and dicotyledons such as cotton, rape, soybean etc., but and only limits to above-mentioned plant.
Learn that from existing result of study at present used resistance glyphosate EPSPS gene all directly clones from plant (comprising rape, petunia, cotton, soybean etc.) and bacterium (comprising intestinal bacteria and Salmonella).The present invention combines the EPSPS proteic different structure territory of plant and bacterial origin and constitutes a kind of EPSPS albumen by redesign.
Existing knowledge shows, the frequency of utilization difference of the codon of coded amino acid in different organisms, and show: the content of G+C is higher in the plant, and other biological body G+C content is lower.The frequency of utilization of this codon directly influences the height of protein expression level.For resistance glyphosate EPSPS albumen can be efficiently expressed in plant, use deflection to be optimized according to plant efficient expressing gene codon the codon in the EPSPS gene order, the content of the 3rd bit base G+C brings up to 68.5%.
At present, the method that obtains gene can be divided into two kinds basically, and a kind of is directly to clone from organism, and another is exactly the method that adopts synthetic.The feature of method for synthesizing gene of the present invention is: the dna double chain is divided into several oligonucleotide fragments carries out artificial chemosynthesis, make these oligonucleotide fragment annealing then, pairing becomes complete double chain DNA molecule.
Existing knowledge shows have two approach can make the resistance of plant acquisition to herbicide glyphosate.A kind of is to make overexpression wild-type EPSPS albumen in the plant materials, and another kind is to make on the wild-type EPSPS albumen to change with glyphosate bonded structural domain, and glyphosate just can not produce restraining effect to it like this.The present invention introduces point mutation according to existing knowledge in EPSPS albumen, make glyphosate can not produce restraining effect to EPSPS albumen, and therefore, described EPSPS has the feature of resistance glyphosate.
EPSPS gene described in the present invention can utilize different method for transformation to be transferred in the plant, and these method for transformation comprise agrobacterium-mediated transformation, pollen tube passage method and particle bombardment etc.
The EPSPS gene is recombinated through the modification on nucleic acid and protein level.The proteic heterozygote sequence of coding EPSPS that the coding region of this gene is made up of the dna sequence dna that derives from plant and microorganism respectively: it is codon optimized that the gene order of this enzyme of encoding selects deflection to carry out according to plant.Described EPSPS enzyme gene adopts chemical process to carry out synthetic.The 5-enol pyruvoyl-shikimic acid-3-phosphate synthase that is obtained has the feature of antiweed.The 5-enol pyruvoyl-shikimic acid-3-phosphate synthase that is obtained can synthesize in plant.
Effect of the present invention is: have higher expression level by present method synthetic EPSPS gene in plant.As previously explained, because in the design process of EPSPS gene, want in the future plant transformed (comprising paddy rice, corn, rape, soybean, cotton etc.) to have a preference for the codon that uses at this gene artificially and be optimized transformation, therefore when this gene is expressed in vegetable cell (with comparing) without codon optimized EPSPS gene, the mRNA that is transcribed out will efficiently be translated by rrna, consequently EPSPS albumen will have higher content in cell, thereby herbicide glyphosate is had the resistance of higher level.
Below with reference to the accompanying drawings embodiments of the invention are described in detail:
Fig. 1 Antiglyphosate gene sequence and amino acid sequence coded thereof,
The segmental synthetic synoptic diagram of Fig. 2 Antiglyphosate gene,
The clone of Fig. 3 Antiglyphosate gene CHEP-1
As shown in Figure 1, the proteic nucleotide sequence of above-listed presentation code EPSPS among the figure, totally 1505 bases, wherein latter two codon is a terminator codon; The amino acid that following expression is coded, is represented with monocase totally by 500.
As shown in Figure 2, with the EPSPS gene separated into two parts of total length, each part is made up of 26 oligonucleotide fragments.The mutual in twos portion paired of these oligonucleotide fragments.
Embodiment:
The design of example 1 EPSPS gene
The design of present embodiment explanation Antiglyphosate gene and the feature of gene itself.Designed full length gene 1506bP, 500 amino acid of encoding altogether, it is TAA TGA that 3 ' end has 2 terminator codons.This gene is a heterozygous genes, the signal peptide in 1～71 amino acids coded plant petunia source, and its effect is that the maturation protein orientation with its back imports in the chloroplast(id) of plant; The part of 72～99 amino acids coding petunia EPSPS n-end of albumen; The proteic part of 100～227 amino acids coding Salmonella (Salmonellatyphimurium) EPSPS; The proteic part of 228～500 amino acids coding intestinal bacteria (E.coli) EPSPS.For this gene can be efficiently expressed in plant, the codon in the gene order is optimized, make the content of the 3rd bit base G+C reach 68.5%.Gene order and coded amino acid are seen Fig. 1.
The chemosynthesis of example 2 EPSPS genes
The purpose of present embodiment is synthetic method and the process of explanation Antiglyphosate gene CHEP-1.Adopt double-stranded synthesis method.With the 744th base A is the boundary, and whole gene is divided into AB two portions, and 5 ' end of A part is designed to the cohesive end of NcoI, and 3 ' end is designed to the cohesive end of PstI; 5 ' overhang of B part is PstI, 3 ' hold cohesive end into SalI.It is 26 oligonucleotide fragments that two chains of A part are divided into, respectively called after A
1A
26Each fragment length is 60bP; The B part is also done same the processing, and each fragment is called after B respectively
1B
26, synthesized the affair oligonucleotide fragment with automatic dna synthesizer after, respectively get 0.2 μ g and in 20 μ l reaction systems, its 5 ' end carried out the phosphoric acid mark with T4 DNA kinases, 37 ℃ of labeled reactants are after 30 minutes, with A
1To A
26Be mixed in an other pipe at 1: 1 by mole ratio, use phenol/chloroform then respectively, chloroform with two kinds of mixture extractings once, add 1/10 volume 3mol/lNaAc (pH5.2) and 2.5 times of volume dehydrated alcohols, placed 30 minutes for-20 ℃, after centrifugal DNA is dissolved in 1/100 the T4 dna ligase damping fluid, totally 30 μ l.Two mixtures were kept 5 minutes in water-soluble at 90 ℃, naturally cool to room temperature then.Like this, A
1～A
26Mixture annealing forms CHEP-1A, B
1～B
26Mixture annealing forms CHEP-1B (Fig. 2).
The clone of example 3 EPSPS genes
Plasmid pUCNH is cut with restriction enzyme NcoI and PstI enzyme, reclaims then, the DNA that reclaims is soluble in water, quantitatively after, mix adding T at 1: 3 by mole ratio with fragment CHEP-1A
4Dna ligase, 16 ℃ of connections are spent the night, transformed into escherichia coli DH5 α, and identify and select positive recombinant.After this positive recombinant cut with PstI and SalI enzyme, fragment CHEP-1B is inserted between these two sites, has just obtained containing the recombinant plasmid of complete genome CHEP-1, pCHEP-1 (Fig. 3).
Claims (7)
1. the method for brand-new synthetic 5-enol pyruvoyl-shikimic acid-3-phosphate synthase gene is characterized in that: this gene process modification, reorganization on Nucleotide and protein level.
2. the method for claim 1, it is characterized in that: said 5-enol pyruvoyl-shikimic acid-3-phosphate synthase gene, the proteic heterozygote sequence of coding EPSPS that the coding region of this gene is made up of the dna sequence dna that derives from plant and microorganism respectively; It is codon optimized that the gene order of this enzyme of encoding selects deflection to carry out according to plant.
3. method as claimed in claim 2 is characterized in that: described 5-enol pyruvoyl-shikimic acid-3-phosphate synthase gene adopts chemical process to carry out synthetic.
4. method as claimed in claim 3 is characterized in that: the 5-enol pyruvoyl-shikimic acid-3-phosphate synthase that is obtained has the feature of antiweed glyphosate.
5. method as claimed in claim 4 is characterized in that: the 5-enol pyruvoyl-shikimic acid-3-phosphate synthase that is obtained can synthesize in plant.
6. method as claimed in claim 5 is characterized in that: said plant comprises dicotyledons such as cotton, soybean, rape, and monocotyledons such as corn, paddy rice, wheat.
7. method as claimed in claim 6 is characterized in that: the method that described plant is transformed can be agrobacterium-mediated transformation, pollen tube passage method and particle bombardment etc.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100404670C (en) * | 2005-01-26 | 2008-07-23 | 中国科学院遗传与发育生物学研究所 | Rice EPSP synthase mutant and its coding gene, obtaining method and application |
| CN1789412B (en) * | 2004-12-16 | 2011-04-20 | 北京大学 | Reconstruction of 5-enol pyruvoyl shikimic acid-3-phosphate synthetase activity by utilizing fragment complementation technique |
| CN103409445A (en) * | 2013-08-01 | 2013-11-27 | 刘坚 | Glyphosate-resistance gene MTP-SMG2-EPSPS and application thereof in cultivation of glyphosate-resistance corn |
-
1999
- 1999-03-31 CN CN 99103472 patent/CN1268572A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1789412B (en) * | 2004-12-16 | 2011-04-20 | 北京大学 | Reconstruction of 5-enol pyruvoyl shikimic acid-3-phosphate synthetase activity by utilizing fragment complementation technique |
| CN100404670C (en) * | 2005-01-26 | 2008-07-23 | 中国科学院遗传与发育生物学研究所 | Rice EPSP synthase mutant and its coding gene, obtaining method and application |
| CN103409445A (en) * | 2013-08-01 | 2013-11-27 | 刘坚 | Glyphosate-resistance gene MTP-SMG2-EPSPS and application thereof in cultivation of glyphosate-resistance corn |
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